Mechanisms of factor recruitment at promoters during RNA polymerase II transcription

Abstract

RNA Polymerase II transcription requires the concerted action of a large number of general transcription factors. These factors must assemble at promoters into a preinitiation complex in order to initiate transcription, and must continually reassemble to promote multiple rounds of transcription in a process termed reinitiation. In vivo, the assembly of these factors is frequently blocked by the packaging of DNA into chromatin, a high order protein/DNA structure. Chromatin remodeling factors must therefore be recruited to promoters to allow the transcription machinery to assemble. In this work I have used an in vitro Saccharomyces cerevisiae nuclear extract system to determine mechanisms by which chromatin remodeling factors and transcription factors can be recruited to promoters during transcription initiation and reinitiation. I found that some acidic transcription activators can recruit both the Swi/Snf ATP-dependent chromatin remodeling complex and the SAGA histone acetyltransferase chromatin remodeling complex to DNA. Interestingly, this recruitment occurs independently of the rest of the transcription machinery and promoter sequences. These results explain how activators can be used to target chromatin remodeling factors to specific genes. I also determined that, to form a preinitiaiton complex, transcription factors can be recruited in at least three cooperative steps. Importantly, preinitiation complex formation requires the presence of a large complex of RNA Polymerase II interacting proteins called Mediator. Finally, I isolated a reinitiation intermediate that contains the majority of the general transcription machinery, including Mediator. Since transcription factor recruitment is a rate limiting step in transcription, this result helps to explain why reinitiation occurs more rapidly than initiation. In addition, the reinitiation intermediate is stabilized by some activators, suggesting a new role for activators in stimulating reinitiation. Taken together, this work begins to address how factor assembly is coordinated during RNA Polymerase II transcription.